1. Technical Field of the Invention
The present invention relates to a procedure or technique for evaluating the sensitizing and/or irritant and/or allergenic effects of a given substrate or active agent.
2. Description of the Prior Art
The skin constitutes one most important organ of a living organism, and is acknowledged to be one of the principal active components of the immune defense system.
The natural human epidermis principally comprises three types of cells, i.e., keratinocytes, which make up the vast majority thereof, melanocytes, and Langerhans cells. By virtue of its specific functions, each of these cell types plays a role in the fundamental mechanisms of the skin of an organism. The Langerhans cells are involved in the immune-conferring defenses of the skin. It has long been known to this art that these cells serve a basic function in the host's immune defenses, in particular as the initial barrier to outside attack.
Langerhans cells are derived from the bone marrow and may be characterized by expression of the CD1a antigenic marker (CD1a-positive cells). In the epidermis, the epidermal Langerhans cells are also characterized by the presence of Birbeck granules (Rowden et al., Nature, 268: 247-248 (1977)). They exercise a decisive function in initiating immune responses against antigens that have invaded the skin or which the skin has newly generated. When placed in contact with an allergen, the Langerhans cells migrate to the ganglions, where they trigger specific reactions of the T cells. In this manner, they are thus comparable to the antigen-presentation cells, which are essential to the proper functioning of the T lymphocytes.
Accordingly, the main function of the Langerhans cells is to supply a sensitization signal in the skin's immune response to a wide variety of antigens, including contact allergens, tumoral antigens, and microorganisms. It may be concluded, therefore, that these cells probably play a role in a large number of skin pathologies.
Furthermore, the industry generally, and the cosmetics trade in particular, formulate new compounds into their compositions with increasing frequency. One of the major problems now confronting the industry is thus the evaluation of the adverse effects which these compounds could induce in contact with the skin, in particular as regards contact sensitization. Hence, it is essential to be able to evaluate, simply and rapidly, the sensitizing and/or irritant and/or allergenic potential of such products/compositions.
For ethical reasons, it is apparent that such evaluations cannot be carried out without problems on humans, and, preferably, they should not be conducted on animal models.
The importance and the necessity of having an in vitro model permitting a safe, rapid, reliable, and inexpensive evaluation of the risk-posing products or substrates are self-evident. Such a model would, moreover, present the additional advantage of permitting problem-free evaluation, at least in preliminary studies, of a large number of materials.
One of the principal obstacles to the development of such model resides in the fact that, in the skin, the Langerhans cells have been subjected to a specific differentiation program. WO-A-90/02796 indicates that, in a three-dimensional skin culture system, Langerhans cells isolated from fresh skin samples be added to keratinocyte and melanocyte cultures. This application also explains that it is difficult to grow these cells in culture. It emerges, in fact, that Langerhans cells purified from epidermal samples are CD1a-positive cells deriving from precursors which have matured during their differentiation cycle to the point where these cells contain Birbeck granules. Once isolated, these cells do not develop further in their differentiation cycle and no longer proliferate.
The in vitro culturing of these cells is limited to maintaining them alive, given the inability of these cells to multiply. Indeed, these cells terminate and die without having fulfilled their function. The same phenomenon is observed when the cells are added to a reconstructed skin model. Therefore, the equivalent skin thus obtained, even if it contains Langerhans cells, does not permit evaluation of the sensitizing and/or irritant and/or allergenic potential of a product, since it does not contain active Langerhans cells.